The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
The general idea of producing valuable proteins into milk using transgene technology was suggested early 1986 (EP 264166, WO 88/00239), but only recently the first true transgenic bioreactors were produced: sheep producing human .alpha..sub.1 AT, goats secreting human tPA and pigs producing human protein C (Wright G et al., Biotechnology vol 9, Sep 1991; Ebert, Karl M et al., Biotechnology vol 9, Sep 1991; Velander, William H et al., Proc Natl Acad Sci vol 89, 12003-7, Dec 1992). The results from those transgenic farm animals and numerous rodent studies have shown that the mammary gland is able to perform posttranslational modifications like glycosylations (N- and O-glycosylations), gammacarboxylations, sulfhydryl bondings and to secrete large amounts of recombinant proteins constitutively into milk during lactation.
Milk protein genes are under particularly complex regulation. Transcriptional and translational activity of milk protein genes are regulated by interactions between cells, between cells and extracellular matrix and by hormonally induced factors (Harris S et al., J Reprod Fert (1990) 88, 707-715). However, most gene constructs, including intact milk protein genes, designed for mammary gland specific expression in transgenic female mammals have shown not to be completely tissue specific (Archibald Alan L et al., Proc Natl Acad Sci vol 87, 5178-82, July 1990; Reddy V B et al., Animal Biotechnology, 2(1), 15-29 (1991); Roberts B et al., Gene, 121 (1992) 255-262; Persuy M-A et al., Eur J Biochem 205, 887-893 (1992)). The reasons for the unspecific expression are related to the gene constructs, the recombinant genes may not contain all the necessary regulatory elements needed for high level, mammary gland-specific expression or the clonings may have created novel tissue specificities (Gunzburg, Walter H et al., Mol Endo 1991, 123-133). Surprisingly Grusby (Grusby M J et al., Proc Natl Acad Sci vol 87, 6897-6901, Sep 1990) found .alpha.-, .beta.- and kappa-casein transcripts in mouse cytotoxic T lymphocytes and Maschio (Maschio A et al., Biochem J (1991) 275, 459-467) reported milk protein transcripts in lactating mouse sebaceous glands suggesting that milk protein genes may not be exclusively mammary gland specific.
Milk whey proteins, .alpha.-lactalbumin and .beta.-lactoglobulin, have been found in the serum of dairy cattle. The concentrations were at highest at parturition (0.5-1 mg/l) and stabilized later at levels of 20-150 ng/l (Mao, Franc C et al., J Dairy Sci 74:2952-58 (1991)). Rabbit whey acidic protein have been found also in serum during lactation (Grabowski H et al., J Dairy Sci 74: 4143-4150 (1991)). It is possible that overaccumulation of milk between milkings is partly compensated by the release of milk protein including the transgene product into blood through a leaky mammary epithelium (Stinnakre M-G et al., Animal Biotechnology, 3(2), 245-255 (1992). Carver (Carver A et al., Cytotechnology 9: 77-84, 1992) found by studying the human .alpha..sub.1 AT producing sheep that the transgene product was found in serum of males and females and blood human .alpha..sub.1 AT concentrations increased upto the range of milligrams per liter to grams per liter during the lactations. Transgenic rats expressing human growth hormone in their milk secreted hGH also into their serum and they exihibited some side effects associated with the elevated hGH concentrations (Ninomiya T et al., Molecular Reproduction and Development 37:276-283 (1994)). The inventors of the present invention have also, as will be reported below, observed elevated human erythropoietin concentrations in the serum of transgenic mice designed to produce human erythropoietin in their milk.
To offer a practically applicable alternative to mammalian tissue culture systems it is important that the producer animals remain healthy also during lactation. Human .alpha..sub.1 AT, tPA and protein C are quite harmless as they are found normally in high quantities in production animals. Apparently the production animals of these proteins have remained in good condition. Severe side effects are, however, probable when producing potent polypeptides like growth factors, cytokines or enzymes, if the gene construct is expressed unspecifically in other tissues than mammary gland or if there is a leakage of the product from the milk into the blood serum. Even low serum levels (less than 1 ng/ml) of a potent polypeptide may be sufficient to cause health problems, especially when the secretion during lactations is more or less constitutive.
The production of desired polypeptides as biologically less active or inactive fusion proteins into the milk of mammals to minimize health problems and possible aglaction has not been reported before the date of the present invention.